Modified Project Summary/Abstract Section Endothelial cells (ECs) in blood vessels within brain parenchyma tightly regulate the function of the blood-brain barrier (BBB) and the communication between the vascular system and neighboring neurons to maintain circuitry homeostasis. In a recent study, we elucidated that Ahnak is a novel regulator of depressive behavior and is an endogenous neuronal scaffolder of the S100A10 (p11)/Anxa2 protein complex and L-type voltage-gated calcium channels, both of which have been implicated in the pathophysiology of psychiatric disorders. In addition to neuronal expression, Ahnak is highly expressed in ECs in blood vessels in the brain. However, Ahnak?s role in the ECs in the regulation of BBB function has not yet been investigated. Our preliminary data indicate that EC-specific Ahnak knockout (KO) mice display an antidepressant-like behavioral phenotype. Because ECs are responsible for BBB permeability, we hypothesize that Ahnak deletion and alterations of Ahnak downstream pathways in ECs modulate BBB permeability and thereby affect neuronal circuit activities. We aim to elucidate Ahnak-mediated molecular and functional pathways in the ECs. We also aim to investigate the roles of Ahnak in ECs in the regulation of BBB function and the communication between the vascular system and neural circuits in the ventral hippocampus. First, we will investigate Ahnak downstream genes and pathways in ECs using an EC-selective TRAP (Translating Ribosome Affinity Purification)/RNA-seq approach. By investigating differentially expressed genes in EC-specific Ahnak KO mice (floxed Ahnak mice crossed with Tek-Cre mice) compared to wild type (WT) controls, we aim to identify downstream molecules and Ahnak-mediated functional pathways (Specific Aim 1). Second, we will investigate the effect of EC-specific Ahnak KO on the BBB permeability in the ventral hippocampus using magnetic resonance imaging (MRI) scans, immunohistochemistry, biochemical assays and transmission electron microscopy (Specific Aim 2). Lastly, we will investigate the effect of Ahnak deletion in ECs on neuronal activity in the ventral dentate gyrus (vDG) using high-density silicon probes recordings of WT and EC-in-vDG (EC[vDG])-specific Ahnak KO mice. EC[vDG]-specific Ahnak deletion will be achieved by a CRISPR/Cas9 approach (Specific Aim 3). The outcome of the proposed studies will identify novel molecular factors and mechanisms regulating homeostasis of the ventral hippocampal circuitry.